The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.

The interfacial properties of a new calcium-silicate-based coronal restorative material (Biodentine™) and a glass-ionomer cement (GIC) with dentin have been studied by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), micro-Raman spectroscopy, and two-photon auto-fluorescence and second-harmonic-generation (SHG) imaging. Results indicate the formation of tag-like structures alongside an interfacial layer called the "mineral infiltration zone", where the alkaline caustic effect of the calcium silicate cement's hydration products degrades the collagenous component of the interfacial dentin. This degradation leads to the formation of a porous structure which facilitates the permeation of high concentrations of Ca(2+), OH(-), and CO(3) (2-) ions, leading to increased mineralization in this region. Comparison of the dentin-restorative interfaces shows that there is a dentin-mineral infiltration with the Biodentine, whereas polyacrylic and tartaric acids and their salts characterize the penetration of the GIC. A new type of interfacial interaction, "the mineral infiltration zone", is suggested for these calcium-silicate-based cements.

Calculations of the electric-field enhancements in the vicinity of an illuminated silver tip, modeled using a Drude dielectric response, have been performed using the finite difference time domain method. Tip-induced field enhancements, of application in "apertureless" Raman scanning near-field optical microscopy (SNOM), result from the resonant excitation of plasmons on the metal tip. The sharpness of the plasmon resonance spectrum and the highly localized nature of these modes impose conditions to better exploit tip plasmons in tip-enhanced apertureless SNOM. The effect of tip-to-substrate separation and polarization on the resolution and enhancement are analyzed, with emphasis on the different field components parallel and perpendicular to the substrate.

Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells’ 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells’ reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC’s interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.

ObjectiveSince their introduction, calcium silicate cements have primarily found use as endodontic sealers, due to long setting times. While similar in chemistry, recent variations such as constituent proportions, purities and manufacturing processes mandate a critical understanding of service behavior differences of the new coronal restorative material variants. Of particular relevance to minimally invasive philosophies is the potential for ion supply, from initial hydration to mature set in dental cements. They may be capable of supporting repair and remineralization of dentin left after decay and cavity preparation, following the concepts of ion exchange from glass ionomers.MethodsThis paper reviews the underlying chemistry and interactions of glass ionomer and calcium silicate cements, with dental tissues, concentrating on dentin–restoration interface reactions. We additionally demonstrate a new optical technique, based around high resolution deep tissue, two-photon fluorescence and lifetime imaging, which allows monitoring of undisturbed cement–dentin interface samples behavior over time.ResultsThe local bioactivity of the calcium-silicate based materials has been shown to produce mineralization within the subjacent dentin substrate, extending deep within the tissues. This suggests that the local ion-rich alkaline environment may be more favorable to mineral repair and re-construction, compared with the acidic environs of comparable glass ionomer based materials.SignificanceThe advantages of this potential re-mineralization phenomenon for minimally invasive management of carious dentin are self-evident. There is a clear need to improve the bioactivity of restorative dental materials and these calcium silicate cement systems offer exciting possibilities in realizing this goal.

Fluorescence lifetime imaging (FLIM) represents a key optical technique for imaging proteins and protein interaction in vivo. We review the principles and recent advances in the application of the technique, instrumentation and molecular probe development.

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The feasibility of apertureless scanning near-field Raman microscopy, exploiting the local enhancement in Raman scattering in the vicinity of a silver or gold tip, was investigated. Using the finite difference time domain method we calculated the enhancement of electric field strength, and hence Raman scattering, achieved through the resonant excitation of local modes in the tip. By modelling the frequency-dependent dielectric response of the metal tip we were able to highlight the resonant nature of the tip-enhancement and determine the excitation wavelength required for the strongest electric field enhancement, and hence Raman scattering intensity, which occurs for the excitation of modes localized at the tip apex. It is demonstrated that a peak Raman enhancement of 10 7 -fold should be achievable with <5 nm spatial resolution. We show that surface-enhanced Raman scattering from carbon contamination on a silver or gold tip can be significant. However, we find for a tip of radius of curvature 20 nm that the Raman enhancement should decay totally within 20 nm from the tip. Hence withdrawal of the tip by this distance should lead to the disappearance of the tip-enhanced signal, leaving only that from carbon contamination on the tip itself and the intrinsic signal from the sample.